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Sex Difference in Expression of Autism Candidate Gene Gabrb3 in Mouse Amygdala
A Major Qualifying Project Submitted to the faculty of the Worcester Polytechnic Institute
in partial fulfillment of the requirements for the Degree of Bachelor of Science
by:
___________________________ Briana Dougherty
Date: April 24, 2008
Approved:
___________________________ Dr. Daniel Gibson III, Major Advisor
Table of Contents Table of Contents ................................................................................................................ 2 Acknowledgements ............................................................................................................. 3 Abstract ............................................................................................................................... 4 Table of Figures .................................................................................................................. 5 Table of Tables ................................................................................................................... 6 12
2.32.4
2.52.6
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5 Appendices ................................................................................................................ 24 6.1Appendix A: Gene Table of Mouse Neurotransmitter Receptors and Regulators ... 24
Introduction ................................................................................................................. 7 Materials and Methods .............................................................................................. 10 2.1 Mouse Model .................................................................................................... 10 2.2 RNA Samples.................................................................................................... 10
SuperArray Analysis ......................................................................................... 10 Individual Gene Analysis .................................................................................. 12 2.4.1 cDNA Synthesis ........................................................................................ 12 2.4.2 Real-time PCR .......................................................................................... 13 siRNA Injection ................................................................................................ 14 Cell Culture ....................................................................................................... 14 2.6.1 Unfreezing the Stock ................................................................................. 15 2.6.2 Maintaining Growth .................................................................................. 15 2.6.3 Neuronal Differentiation ........................................................................... 15 2.6.4 siRNA Treatment ...................................................................................... 16 2.6.5 Harvesting Cells ........................................................................................ 17 2.6.6 Freezing Cells ........................................................................................... 18
Results ....................................................................................................................... 19 3.1 SuperArray Results ........................................................................................... 19 3.2 Individual Gene Results .................................................................................... 20 3.3 siRNA Results ................................................................................................... 20
4 Discussion ................................................................................................................. 21 Works Cited .............................................................................................................. 22
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Acknowledgements I would like to thank Dr. Jun Xu of Tufts University Cummings School of
Veterinary Medicine for allowing me the use of his lab and always pushing me in the right direction.
I would also like to thank Dr. Elizabeth Byrnes of Tufts University Cummings School of Veterinary Medicine for her expertise with mouse brains.
Thanks to Dr. Daniel Gibson of Worcester Polytechnic Institute for his support and guidance.
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Abstract This project targeted gene expression in specific brain regions known to play a
role in autism spectrum disorders (ASD). Due to ASD affecting males four times more often than females, we compared the male and female gene expression in different brain regions. Specific brain regions were extracted and analyzed using a PCR panel containing 84 neurotransmitter genes to identify differences between males and females in gene expression. Gabrb3 was found to be significantly higher in females than males. siRNA gene silencing was tested in the second part of the project for studying functionality. In the trial, the injection of gene specific siRNA, Mapk1, was shown to completely silence expression of the targeted gene. This would be the next step in identifying which genes are specific to autism.
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Table of Figures Figure 1: Location of amygdala. ......................................................................................... 7 Figure 2: RNAi pathway. .................................................................................................... 9 Figure 3: RNA integrity can be viewed by gel electrophoresis ........................................ 10 Figure 4: Graph showing linearity of serial dilution. ........................................................ 13 Figure 5: Graph showing dissociation curve ................................................................... 14 Figure 6: D6 retinoic acid treatment ................................................................................ 16 Figure 7: Gabrb3 and Sstr3 Expression ........................................................................... 20 Figure 8: Mapk1-siRNA Expression. ............................................................................... 20
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Table of Tables Table 1: Gene Table of Mouse Neurotransmitter Receptors and Regulators ................. 11
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1 Introduction
Autism spectrum disorders (ASD) are neurodevelopment disorders that are
characterized by impaired social interaction, deficits in communication, and repetitive
behaviors and restricted interests. One out of 166 newborns is affected by ASD, likely
caused by both genes and environment. Parents are usually the first to notice a problem
with the child, usually noticing that the child is unresponsive to people and/or focusing
on one thing intently for an extended period of time (National Institute of Mental Health).
The developmental disorders can range from mild to severe. There is no single best
treatment plan for all affected children. Early intervention is vital since individuals
respond to highly specialized programs.
Genetic factors are known to play a role in the etiology, as indicated by the
difference in concordant rates for ASD between identical twin (ranging from 36% to
96%) and dizygotic twin (0%). The focus has been brought to specific chromosomal
regions that possibly contain autism-related genes (National Alliance for Autism
Research). These “autism susceptible genes” make certain individuals more vulnerable
by influencing their brain development and/or function. One such gene is Gabrb3 which
has an important role in neuronal
communication (see below).
In neuropathologic examinations,
anatomical changes have been consistently
found in autism patients in the cerebellum,
cerebral cortex, and limbic system. Based on
recent technologies, scientists have been able
to study the structure and function of specific
brain regions. Some of these technologies
include positron tomography (PET),
computerized tomography (CT) and
magnetic resonance imaging (MRI). Using these new advances, major brain structures
associated with autism have been found, including the cerebellum, cerebral cortex, corpus
Figure 1: Location of amygdala.
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callosum, brain stem, hippocampus, and the limbic system (National Institute of Mental
Health).
One important component of the limbic system is the amygdala, which was chosen
as the focus of our initial analysis. The amygdala plays an important role in appraising
emotionally relevant information in social interaction. It is also responsible for emotional
responses, including aggressive behavior (National Institute of Mental Health). Altered
neural activities in the amygdala were revealed by fMRI and PET scans in people with
ASD while they were engaged in social perception tasks. In the amygdala, GABAergic
neurons play an essential role in emotion perception and memory formation. Specifically
GABA-A receptor genes including Gabrb3 have been identified as autism loci in genome
scans.
GABAA receptors are important for proliferation, migration, and differentiation of
precursor cells that coordinate development of the embryonic brain. GABAA receptors
consist of eight different types of subunits: α1-6, β1-4, γ1-3, δ, ρ1-2, π, ε, and θ. Protein
subunits co-assemble with other protein molecules to form an oligomeric protein.
Amongst GABA subunit genes, there is a cluster consisting of Gabrb3, Gabra5, and
Gabrg3. Together, the proteins coded by this gene cluster have unique regional and
distribution in the central nervous system. Gabrb3 is vital for both mature brain function
and correct brain development (DeLorey, 208). Based on a study by DeLorey, et. al., it
has been found that gabrb3-/- mice exhibit social deficits in behavior, similar to that of
ASD. This leads us to believe that the gene Gabrb3 has a part to play in the development
of ASD.
Boys are four times more likely to be diagnosed with ASD than girls. The
underlying biological mechanism is unclear. One possibility is that gene expression, and
in turn neuronal activity differs between the two sexes in autism-related brain regions
such as the amygdala. Mice and humans show similar gene expression patterns in the
brain and therefore we performed gene expression analysis in the amygdala between
adult male and female mice.
Once a gene is found to be expressed differently between males and females, its
involvement in amygdala function as well as social behavior can be further analyzed by
RNA interference method, which allows brain-region specific gene silencing.
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RNA interference, or RNAi, is a
mechanism for RNA guided regulation
of gene expression where double
stranded RNA (dsRNA) inhibits the
expression of genes with
complementary nucleotide sequences.
The pathway can be seen in Figure 2.
The RNAi pathway is initiated by
dicer, an enzyme that cleaves dsRNA
to short fragments about 20-25 base-
pairs in length. The guide strand is
incorporated into the RNA-induced
silencing complex, or RISC, where it
base-pairs with its complementary
sequence.
The most well known outcome of this is post-transcriptional RNA gene silencing.
This occurs when the guide strand base-pairs with mRNA to induce degradation of the
mRNA by the catalytic component of the RISC complex. The results of this are short
RNA fragments and are commonly known as small interfering RNA (siRNA) or
microRNA (miRNA). The difference between siRNA and miRNA are dependant upon
whether the fragments are from exogenous sources, siRNA, or when they are produced
endogenously from RNA-coding genes, miRNA.
Figure 2: RNAi pathway. The enzyme dicer trims double stranded RNA, to form small interfering RNA or
microRNA. These processed RNAs are incorporated into the RNA-induced silencing complex (RISC), which
targets messenger RNA to prevent translation (Hammond, S)
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2 Materials and Methods
2.1 Mouse Model
The animals used in this study were two month old adult black 6 mice. The brain
was freshly dissected from a two month old mouse and then frozen. The frozen brain
was then placed in the cryostat and a 1 mm micropunch was used to isolate specific brain
regions such as amygdala.
2.2 RNA Samples
An RNA extraction was performed using
Qiagen’s RNAeasy Mini Kit to isolate the RNA
(Qiagen, Valencia, CA). The protocol followed
was the one detailed in the manual found on the
Qiagen website,
(http://www1.qiagen.com/literature/handbooks/l
iterature.aspx?id=1000291). The total RNA
obtained was then run in the spectrophotometer
to measure the concentration of the RNA. A
1.5% agarose gel was also run at 90 volts to
assess the quality of the RNA. The quality of
the RNA was confirmed by the presence of two bands, 18S and 28S bands which can be
seen in Figure 3.
Figure 3: RNA integrity can be viewed by gel electrophoresis
2.3 SuperArray Analysis
Neurotransmitter Receptor SuperArrays (SuperArray, Frederick, MD) were used
which are 96-well plates allowing simultaneous measurements of 84 genes on a real time
PCR machine. The protocol followed was the one that was detailed in the manual online,
(http://www.superarray.com/Manual/pcrarrayplate.pdf). These genes encode receptors
for neurotransmitters such as acetyl-choline, dopamine, and GABA. Four pooled
samples, 2 male and 2 female samples, were tested. Each sample contained amygdala
RNA derived from 3 mice.
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Ache
A01
Anxa9
A02
Brs3
A03
Cckar
A04
Cckbr
A05
Chat
A06
Chrm1
A07
Chrm2
A08
Chrm3
A09
Chrm4
A10
Chrm5
A11
Chrna1
A12
Chrna2
B01
Chrna3
B02
Chrna4
B03
Chrna5
B04
Chrna6
B05
Chrna7
B06
Chrnb1
B07
Chrnb2
B08
Chrnb3
B09
Chrnb4
B10
Chrnd
B11
Chrne
B12
Chrng
C01
Comt
C02
Drd1a
C03
Drd2
C04
Drd3
C05
Drd4
C06
Drd5
C07
Gabra1
C08
Gabra2
C09
Gabra3
C10
Gabra4
C11
Gabra5
C12
Gabra6
D01
Gabrb2
D02
Gabrb3
D03
Gabrd
D04
Gabrg1
D05
Gabrg2
D06
Gabrp
D07
Gabrq
D08
Gabrr1
D09
Gabrr2
D10
Gad1
D11
Galr1
D12
Galr2
E01
Galr3
E02
Glra1
E03
Glra2
E04
Glra3
E05
Glra4
E06
Glrb
E07
Gpr103
E08
Npffr1
E09
Prokr1
E10
Prokr2
E11
Npffr2
E12
Gpr83
F01
Grpr
F02
Htr3a
F03
Maoa
F04
Mc2r
F05
Nmur1
F06
Nmur2
F07
Npy1r
F08
Npy2r
F09
Npy5r
F10
Npy6r
F11
Ntsr1
F12
Pgr15l
G01
Ppyr1
G02
Prlhr
G03
Slc5a7
G04
Sstr1
G05
Sstr2
G06
Sstr3
G07
Sstr4
G08
Sstr5
G09
Tacr1
G10
Tacr2
G11
Tacr3
G12
Gusb
H01
Hprt1
H02
Hsp90
ab1
H03
Gapdh
H04
Actb
H05
MGDC
H06
RTC
H07
RTC
H08
RTC
H09
PPC
H10
PPC
H11
PPC
H12
Table 1: Gene Table of Mouse Neurotransmitter Receptors and Regulators
A detailed list of the genes can be found in Appendix A on page 24 of this document.
Expression of these genes was shown based on comparative expression of Gapdh
expression. These results were calculated using the following calculations.
The relative expression of each gene was calculated using the formula:
ΔCt = CtGOI – Ct AVG(HKG)
Where CtGOI is the Ct value of gene of interest determined on the real time PCR
machine indicating abundance of specific mRNA and CtAVG(HKG) is the average Ct
value of five housekeeping genes.
The relative expression (Exp) of each gene was calculated using the formula,
Exp = 2-ΔCt (ΔCt = CtGOI – CtGapdh)
To determine statistical significance (p < .05) t-tests were used to compare males and
females. Six samples were included for each sex.
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The SuperArray kit simultaneously measures 84 genes in a 96 well plate. The
remaining 12 wells contain a combination of controls. Wells H1-H5 contain the
housekeeping genes. These genes are control genes that should definitely show
expression. These genes include: Gusb, Hsprt1, Hsp90ab1, Gapdh, and Actb. The
housekeeping genes are used to normalize expression in the other 84 genes.
Another important gene of the control genes is the MGDC, or mouse genomic
DNA contamination. It can be found in well H6. Since there is no purification method
that can guarantee that RNA is completely DNA free, it is vital to know if there is any
residual DNA that is still present with the RNA. The MGDC tests specifically for
genomic DNA contamination in each sample. A MGDC threshold cycle less than 30
indicates a significant presence of genomic DNA contamination.
2.4 Individual Gene Analysis
2.4.1 cDNA Synthesis
Complementary DNA (cDNA) was synthesized from RNA. When synthesizing
cDNA, it is necessary to remove any DNA contaminants. This was done with a genomic
DNA elimination step. In order to make DNA-free RNA, a kit by Ambion (Austin, TX)
was used. The protocol followed was the one given by the company
(http://www.ambion.com/techlib/prot/bp_1906.pdf).
Using the DNA free RNA, cDNA was synthesized using Invitrogen’s SuperScript
III Platinum Two-Step qRT-PCR Kit (Invitrogen, Valencia, CA). The protocol followed
was the one detailed in the manual. The cDNA synthesized was later used to test specific
gene primers to identify any sex differences using real-time PCR.
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2.4.2 Real-time PCR
cDNA was synthesized with reverse transcriptase (Invitrogen) followed by real
time PCR quantifications using gene-specific primers. Primer sequences were obtained
from the PrimerBank website and primers were ordered from IDT (Coralville, IA).
Figure 4: Graph showing linearity of serial dilution.
Each primer pair was tested for linearity with serial diluted samples and
specificity which was indicated by aligned disassociation curves across samples. Gapdh
expression was also quantified serving as a reference for loading.
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Figure 5: Graph showing dissociation curve
2.5 siRNA Injection
Gene specific siRNA was purchased from Qiagen and mixed with DOTAP (Roche,
Penzberg, Germany) which served as transfection vector. Each injection of 3.75μg
siRNA in 1μl was delivered with a Precision pump into the brain of adult BL6 mice.
Brain region specific expression of the target gene was analyzed two days after the
injection.
2.6 Cell Culture
P19 stem cells were used for the cell cultures. Three different plates were used,
NUNC plates, Falcon plates, and 12-well plates. The NUNC plates were used for general
growth of stem cells. The Falcon plates were used for retinoic acid-induced neuronal
differentiation. The 12-well plates were used for siRNA treatments. The medium used
for maintaining cells was Dulbecco’s Modified Eagle’s Medium (DMEM) containing
10% fetal bovine serum and 1% antibiotic. Phosphate Buffer Saline was used to wash the
cells and trypsin was used to detach the cells. The cells were grown and stored in a 37°C
incubator.
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2.6.1 Unfreezing the Stock
The stock was frozen in 1 ml of Freezing Medium which is toxic to the cells. In
order to prevent cell death, the stock is only partially thawed. When the stock was
partially thawed, it was dumped into a 15 ml conical tube containing 10 ml DMEM. The
conical tube was then inverted until everything was mixed. The cells were then
centrifuged for five minutes. After five minutes, the cells form a pellet at the bottom of
the conical tube. The excess medium was then removed and the pellet was resuspended
in 2ml of DMEM. The cells were then plated in 10 ml DMEM in a NUNC plate.
2.6.2 Maintaining Growth
The protocol for maintaining growth is pretty consistent among cell culture labs.
Once the cells are plated in a NUNC plate they were incubated at 37°C for two days.
After the two day incubation period, the DMEM was removed and 5 ml of PBS was
added to wash the cells. The PBS was gently swirled and then removed. Then, 1 ml of
trypsin was added to the plate to detach the cells from the bottom of the plate. Once the
trypsin was added, the plate was placed in the 37°C incubator for three minutes. The
plate was gently hit to detach the cells from the plate. In order to stop the trypsinization,
9 ml of DMEM was added to the plate. In order to break up the cells, it was necessary to
pipette up and down about ten times. A new plate was labeled and 10 ml of DMEM was
placed in the plate. Only 0.5 ml of cells were placed in 10 ml of DMEM leaving us with
a 1:20 dilution. The cells were then incubated for two days at 37°C.
2.6.3 Neuronal Differentiation
Retinoic acid (RA) treatment induces P19 stem cells to differentiate into neurons.
The RA treated cells were plated in Falcon plates, where the cells did not adhere to the
bottom of the plate. The cells were still incubated for 2 days at 37°C.
After stopping trypsinization of the cells, 1.5 – 3 ml of cells were plated in a
Falcon plate containing 10 ml DMEM and 3 µl RA/ 10 ml medium. The plate was then
stored at 37°C, this is neuronal differentiation Day 1 (D1). Cells form aggregates in
suspension.
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After two days, the medium was changed by removing all the media from the
plate and transferring it to a 15 ml conical tube. The tube was left to sit for five minutes
to allow the cells to settle to the bottom of the tube. During this time, 10 ml DMEM and
3 µl RA were mixed in a new tube. The medium was then removed and the fresh
DMEM/RA mixture was used to resuspend the cells. The total volume was transferred to
a new Falcon plate and incubated for another two days at 37°C.
On D5, RA treatment is complete. The total volume from the plate is removed
and put into a 15 ml conical tube and allowed to sit for five minutes so the cells will settle
at the bottom. The medium was removed from the tube and washed with 5 ml PBS,
dispersed by flicking tube. After washing, the PBS was removed and 3 ml of trypsin was
added and dispersed by flicking the tube.
The tube was then incubated for five
minutes at 37°C. After five minutes, the
trypsin was removed, as much as possible,
and 2 ml of medium was added. By
pipetting up and down about ten times to be
sure the cells were completely dispersed. 1
ml of cells was plated in 10 ml DMEM in
NUNC a plate.
On D7, Ara-C treatment was started.
Ara-C incorporates into the DNA in dividing cells and thus kills any cells that divide
(such as glia and fibroblasts as well as undifferentiated stem cells). Therefore, Ara-C
kills any stem cells that were not differentiated into neurons. For 1 ml of DMEM, 1 µl of
Ara-C is needed. The DMEM present in the plate was removed and 10 ml DMEM plus
10 µl Ara-C were then added gently to the plate. The cells were incubated for two days
at 37°C.
Neuron
Figure 6: D6 retinoic acid treatment
2.6.4 siRNA Treatment
siRNA treatment allows for a knockdown of a specific gene of interest. As a trial,
we tested knockdowns in mitogen activated protein kinase 1 (MAPK1) and Jarid1c as the
gene of interest. Jarid1c mutations have been found to have a part in both X-linked
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mental retardation as well as aggression. MAPK1-siRNA is a positive control sold by
Qiagen.
Shortly before transfection occurred, 100 µl of cells were seeded in each well of a
12-well plate in 1 ml DMEM. The plate was then incubated under normal growth
conditions (37°C). Then, 100 µl of DMEM without FBS was mixed with 3 µl of 2 µM
siRNA and 6 µl HiPerfect Transfection Reagent (Qiagen) by vortexing. The mixture was
then incubated at room temperature for 5-10 minutes to allow a complex to form. The
siRNA was specific for one of three genes, MAPK1, Jarid1c, or a negative control. The
complex that formed was added into the wells containing newly seeded cells in a drop-
wise fashion, and after gently rocking the plate to ensure uniform distribution; the plate
was placed back into the 37°C incubator for two days.
After two days, a photo was taken of each well and then they were harvested. The
DMEM was removed and each well was washed with 1 ml of PBS. The PBS was
removed and 500 µl of RLT + 5 µl βME were added to each well. It was allowed to sit
for two minutes and then the plate was struck to disrupt the cells. Everything was
completely removed from each well and placed in a labeled microcentrifuge tube and
stored at -20°C. These cells were later used to extract RNA from.
2.6.5 Harvesting Cells
Harvesting the cells allows us to freeze for later use, most likely an RNA
extraction.
The DMEM was removed from the plate. The cells were washed with 5 ml of
PBS, and the PBS was gently removed. The cells were trypsinized using 1 ml of trypsin,
incubating the plate for two minutes at 37°C. The trypsinization process was stopped
with 2 ml of DMEM. As long as the cell types are the same, they can be combined into
one tube and then centrifuged for five minutes. Then the DMEM + trypsin was removed
from the cells and washed with 5 ml PBS by disrupting the pellet. Centrifuge again for
five minutes. Removing as much of the supernatant as possible, the cell were then ready
to be stored at -80°C.
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2.6.6 Freezing Cells
Freezing cells is helpful if you are going to be away from the cells for more than a
few days. The DMEM was removed from the plate. The cells were washed with 5 ml of
PBS and then the PBS was gently removed. The cells were trypsinized using 1 ml
trypsin, incubating the plate for three minutes. The cells were dispersed by hitting the
plate. The trypsinization was stopped by adding 5 ml DMEM. Then, the tube was
centrifuged for five minutes. Without disturbing the pellet, as much medium as possible
was removed. The pellet was then resuspended in freezing medium containing DMSO
and 1 ml of the mixture was added to each cryotube. The cells were then stored at -80°C.
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3 Results
3.1 SuperArray Results
SuperArray analyses of 84 neurotransmitter receptor genes were performed on 4
biologically independent samples (2 per sex). Some genes were found to have higher
expression in females such as Sstr3 and Gabrb3, and some higher in males, such as
Anxa9. Gabrb3 is a receptor subunit for the neurotransmitter GABA, a major inhibitory
transmitter of the nervous system. Mutations in this gene may be associated with the
pathogenesis of Angelman syndrome, Prader-Willi syndrome, and autism. (National
Center for Biotechnology Information, Gabrb3). Sstr3 is somatosation receptor 3. It acts
to inhibit the release of hormones and other secretory proteins at many different sites. It
is expressed in the highest levels in brain and pancreatic islets. (National Center for
Biotechnology Information, Sstr3). Anxa9 (annexin A9) encodes a divergent member of
the annexin protein family having a structural analysis that suggests the conserved
putative ion channel that is formed by the tetrad core. (National Center for
Biotechnology, Anxa9).
In addition, some genes were found to be more actively transcribed in the
amygdala, Gad1 and Gabra1, while some were expressed at much lower levels, Npy6r
and Tacr2. Gad1, glutatmate decarboxylase 1, is the rate-limiting enzyme in GABA
synthesis and is identified as a major autoantigen in insulin-dependant diabetes and
deficiency in this enzyme has been shown to lead to seizures (National Center for
Biotechnology Information, Gad1). Like Gabrb3, Gabra1 is a receptor subunit for the
major inhibitory neurotransmitter GABA in the mammalian brain. At least 16 distinct
subunits of GABA-A receptors have been identified (National Center for Biotechnology
Information, Gabra1). Tacr2, tachykinin receptor 2, belongs to a family of genes that
functions as receptors belonging to this family are characterized with G proteins and 7
hydrophobic transmembrane regions.
Our results such as the female-bias in expression of Sstr3 are consistent with
previous findings including the up-regulatory effect of estrogens on Sstr3 expression.
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3.2 Individual Gene Results
Gabrb3 and Sstr3 were verified to be expressed more highly in the female
amygdala than in males (*: p < .05 in both cases). Another GABA receptor gene,
Gabra5, showed no sex difference in expression therefore the sex difference appears to
be specific for certain GABA receptor genes but not others. No sex difference was found
in expression of Anxa9 when 6 male and 6 female amygdala samples were tested, in spite
of the SuperArray result.
Figure 7: Graph A compares expression of Gabrb3 between males and females which shows that females had a higher level of Gabrb3 mRNA in the amygdala than males. Graph B shows that the female bias in expression is consistent with up-regulatory effects of estrogen on Sstr3 expression.
Figure 7A shows the difference in Gabrb3 expression. As it can be seen, the expression
of Gabrb3 shows a significant difference, * = p < 0.5 with a higher expression in females.
Figure 7B shows the difference in Sstr3 expression, and it also shows a significant
difference, * = p < 0.5.
3.3 siRNA Results
As a test for methodology,
siRNA specific for Mapk1 was
injected into the dorsal
hippocampus and quantitative real
time PCR was performed 48 hr
later. Mapk1 was found to be specifically
silenced as can be seen in Figure 8.
Figure 8: Expression of Mapk1 siRNA implying that Mapk1-siRNA treated brains the expression of Mapk1 was silenced.
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4 Discussion
Gabrb3, a gene which is implicated in autism in humans, was found to be expressed
more highly in female amygdala than in males in mice. It is unclear whether a similar
sex difference will be identified in humans and whether such a sex difference contributes
to the difference in vulnerability in autism. If Gabrb3 is also expressed more highly in
human females than males, we can make the connection that Gabrb3 has some affect on
blocking autism in humans. In the future we will compare Gabrb3 expression in BTBR
mice. BTBR mice are so called “autistic mice”. These mice exhibit autistic physical
behaviors, such as anti-social behavior, and not playing as much with other mice. In
addition, using siRNA brain region-specific gene silencing, we will delineate in the future
whether Gabrb3 gene plays a role in sex differences in behavior such as mouse social
play behavior. Our goal for the future would be to inject Gabrb3 siRNA into the
amygdala.
Sstr3, a gene encoding somatosatin receptor subtype, was also found to be expressed
higher in females than in males. This sex difference has been previously shown to be due
to the effect of estrogen. Based on this information we cannot deduce what effect Sstr3
has regarding autism. The next step would be to do comparative analysis between male
and female mice, as well as between males with no testes and females with testosterone.
Based on our findings, we cannot make any definitive conclusions on what genes
affect human autism. However, this evidence will hopefully lead us in the correct
direction to isolate the genes responsible for autism in humans.
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5 Works Cited
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Baron-Cohen, Simon, Rebecca C. Knickmeyer, and Matthew K. Belmonte. "Sex Differences in the Brain: Implications for Explaining Autism." Science 310 (2005): 819.
Bolivar, Valerie J., Samantha R. Walters, and Jennifer L. Phoenix. "Assessing Autism-Like Behavior in Mice: Variations in Social Interactions among Inbred Strains." Behavioral Brain Research 176 (2007): 21-6.
DeLorey, Timothy M., et al. "Gabrb3 Gene Deficient Mice Exhibit Impaired Social and Exploratory Behaviors, Deficit in Non-Selective Attention and Hypoplasia of Cerebellar Vermal Lobules: A Potential of Autism Spectrum Disorder." Behavioral Brain Research 187 (2008): 207-20.
Hammond S, Bernstein E, Beach D, Hannon G (2000). "An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells". Nature 404 (6775): 293–6. PMID 10749213.
National Alliance for Autism Research. "Exploring Autism." Brilliant Content. 2002. <http://www.exploringautism.org/autism/index.htm>.
National Center for Biotechnology Information. "Entrez Gene: Anxa9." March 13 2008. <http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=8416>.
---. "Entrez Gene: Gabra1." April 6 2008. <http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=2554>.
---. "Entrez Gene: Gabrb3." April 13 2008. <http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=2562>.
---. "Entrez Gene: Gad1." April 13 2008. <http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=2571>.
---. "Entrez Gene: Sstr3." April 6 2008. <http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=6753>.
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National Institute of Child Health and Human Development. "Autism Overview: What We Know." National Institute of Health. May 2005. <http://www.nichd.nih.gov/publications/pubs/upload/autism_overview_2005.pdf>.
National Institute of Mental Health. "Autism Spectrum Disorders." National Institute of Health. April 3 2008. <http://www.nimh.nih.gov/health/publications/autism/complete-publication.shtml>.
Pachernik, J., et al. "Retnoic Acid-Induced Neural Differentiation of P19 Embryonal Carcinoma Cells is Potentiated by Leukemia Inhibitory Factor." Physiological Research (2005) . April 10, 2008 <http://www.biomed.cas.cz/physiolres/pdf/54/54_257.pdf>.
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6 Appendices
6.1 Appendix A: Gene Table of Mouse Neurotransmitter Receptors and Regulators
Position Symbol Description
A01 Ache Acetylcholinesterase
A02 Anxa9 Annexin A9
A03 Brs3 Bombesin-like receptor 3
A04 Cckar Cholecystokinin A receptor
A05 Cckbr Cholecystokinin B receptor
A06 Chat Choline acetyltransferase
A07 Chrm1 Cholinergic receptor, muscarinic 1, CNS
A08 Chrm2 Cholinergic receptor, muscarinic 2, cardiac
A09 Chrm3 Cholinergic receptor, muscarinic 3, cardiac
A10 Chrm4 Cholinergic receptor, muscarinic 4
A11 Chrm5 Cholinergic receptor, muscarinic 5
A12 Chrna1 Cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle)
B01 Chrna2 Cholinergic receptor, nicotinic, alpha polypeptide 2 (neuronal)
B02 Chrna3 Cholinergic receptor, nicotinic, alpha polypeptide 3
B03 Chrna4 Cholinergic receptor, nicotinic, alpha polypeptide 4
B04 Chrna5 Cholinergic receptor, nicotinic, alpha polypeptide 5
B05 Chrna6 Cholinergic receptor, nicotinic, alpha polypeptide 6
B06 Chrna7 Cholinergic receptor, nicotinic, alpha polypeptide 7
B07 Chrnb1 Cholinergic receptor, nicotinic, beta polypeptide 1 (muscle)
B08 Chrnb2 Cholinergic receptor, nicotinic, beta polypeptide 2 (neuronal)
B09 Chrnb3 Cholinergic receptor, nicotinic, beta polypeptide 3
B10 Chrnb4 Cholinergic receptor, nicotinic, beta polypeptide 4
B11 Chrnd Cholinergic receptor, nicotinic, delta polypeptide
B12 Chrne Cholinergic receptor, nicotinic, epsilon polypeptide
C01 Chrng Cholinergic receptor, nicotinic, gamma polypeptide
C02 Comt Catechol-O-methyltransferase
C03 Drd1a Dopamine receptor D1A
C04 Drd2 Dopamine receptor 2
C05 Drd3 Dopamine receptor 3
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C06 Drd4 Dopamine receptor 4
C07 Drd5 Dopamine receptor 5
C08 Gabra1 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 1
C09 Gabra2 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 2
C10 Gabra3 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 3
C11 Gabra4 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 4
C12 Gabra5 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 5
D01 Gabra6 Gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 6
D02 Gabrb2 Gamma-aminobutyric acid (GABA-A) receptor, subunit beta 2
D03 Gabrb3 Gamma-aminobutyric acid (GABA-A) receptor, subunit beta 3
D04 Gabrd Gamma-aminobutyric acid (GABA-A) receptor, subunit delta
D05 Gabrg1 Gamma-aminobutyric acid (GABA-A) receptor, subunit gamma 1
D06 Gabrg2 Gamma-aminobutyric acid (GABA-A) receptor, subunit gamma 2
D07 Gabrp Gamma-aminobutyric acid (GABA-A) receptor, pi
D08 Gabrq Gamma-aminobutyric acid (GABA-A) receptor, subunit theta
D09 Gabrr1 Gamma-aminobutyric acid (GABA-C) receptor, subunit rho 1
D10 Gabrr2 Gamma-aminobutyric acid (GABA-C) receptor, subunit rho 2
D11 Gad1 Glutamic acid decarboxylase 1
D12 Galr1 Galanin receptor 1
E01 Galr2 Galanin receptor 2
E02 Galr3 Galanin receptor 3
E03 Glra1 Glycine receptor, alpha 1 subunit
E04 Glra2 Glycine receptor, alpha 2 subunit
E05 Glra3 Glycine receptor, alpha 3 subunit
E06 Glra4 Glycine receptor, alpha 4 subunit
E07 Glrb Glycine receptor, beta subunit
E08 Gpr103 G protein-coupled receptor 103
E09 Npffr1 Neuropeptide FF receptor 1
E10 Prokr1 Prokineticin receptor 1
E11 Prokr2 Prokineticin receptor 2
E12 Npffr2 Neuropeptide FF receptor 2
F01 Gpr83 G protein-coupled receptor 83
F02 Grpr Gastrin releasing peptide receptor
F03 Htr3a 5-hydroxytryptamine (serotonin) receptor 3A
F04 Maoa Monoamine oxidase A
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F05 Mc2r Melanocortin 2 receptor
F06 Nmur1 Neuromedin U receptor 1
F07 Nmur2 Neuromedin U receptor 2
F08 Npy1r Neuropeptide Y receptor Y1
F09 Npy2r Neuropeptide Y receptor Y2
F10 Npy5r Neuropeptide Y receptor Y5
F11 Npy6r Neuropeptide Y receptor Y6
F12 Ntsr1 Neurotensin receptor 1
G01 Pgr15l G protein-coupled receptor 15-like
G02 Ppyr1 Pancreatic polypeptide receptor 1
G03 Prlhr Prolactin releasing hormone receptor
G04 Slc5a7 Solute carrier family 5 (choline transporter), member 7
G05 Sstr1 Somatostatin receptor 1
G06 Sstr2 Somatostatin receptor 2
G07 Sstr3 Somatostatin receptor 3
G08 Sstr4 Somatostatin receptor 4
G09 Sstr5 Somatostatin receptor 5
G10 Tacr1 Tachykinin receptor 1
G11 Tacr2 Tachykinin receptor 2
G12 Tacr3 Tachykinin receptor 3
H01 Gusb Glucuronidase, beta
H02 Hprt1 Hypoxanthine guanine phosphoribosyl transferase 1
H03 Hsp90ab1 Heat shock protein 90kDa alpha (cytosolic), class B member 1
H04 Gapdh Glyceraldehyde-3-phosphate dehydrogenase
H05 Actb Actin, beta, cytoplasmic
H06 MGDC Mouse Genomic DNA Contamination
H07 RTC Reverse Transcription Control
H08 RTC Reverse Transcription Control
H09 RTC Reverse Transcription Control
H10 PPC Positive PCR Control
H11 PPC Positive PCR Control
H12 PPC Positive PCR Control